2.1. Introduction
Biopharmaceuticals are a very important class of therapeutics for the treatment of a vast array of diseases. These products, however, are very expensive and thus unaffordable by most of the population both in the U.S. and worldwide. Much of the high cost is due to the purification processes (downstream processing), which account for over 50% of the overall manufacturing costs. A. C. A. Roque, C. S. O. Lowe and M. A. Taipa, Biotechnol. Prog. 20 (2004) 639-654.
Chromatography is central in downstream processing of biologics, as it is a scalable technique that has the ability to achieve the high purity standards required by the regulatory authorities for commercial bioproducts. G. Sofer, J. Chromatogr. A 707 (1995) 23. Among the different types of chromatography, affinity chromatography has a very high potential for the selective purification of therapeutic proteins. In order to be applied on the industrial scale, an affinity adsorbent should be characterized by high binding capacity and specificity, low cost and high chemical resistance toward the treatments of cleaning-in-place (CIP) and sanitization-in-place (SIP) periodically performed to ensure its safe reusability. G. Jagschies, Process-Scale Chromatography, Ullmann's Encyclopedia of Industrial Chemistry (2000). The industrial standard for CIP and SIP of chromatographic media is aqueous NaOH (0.1M-1M), as it is the most cost effective agent in eliminating bacteria, endotoxins, viruses and does not present any environmental disposal problems. M. Linhult, S. Gulich, S. Hober, Protein Pept. Lett. 12 (2005) 305; GE Healthcare Application Note: Use of sodium hydroxide for cleaning and sanitizing chromatography media and systems. (Code No. 18-1124-57 AG). However, to date, most widely used affinity ligands suffer from chemical instability towards these procedures.
Among the therapeutic proteins, monoclonal antibodies (mAbs) are an important class of biologics for the treatment of diseases such as cancer, skin disorders, neurological disorders and other autoimmune diseases. They account for 43% of total market value of therapeutic proteins. Strohl W. R. and Knight D. M., (2009) Discovery and development of biopharmaceuticals: current issues. Curr. Opin. Biotechnol. 20, 668-672. However, the manufacture of mAb-based biologics is affected by several drawbacks related to the use of Protein A and Protein G affinity chromatography. First, the high price of Protein A and Protein G affinity media impacts significantly on the overall manufacturing costs. Also, these media bind very strongly to the antibodies, thus requiring the application of harsh elution conditions, which can result in reduced activity and/or yield of the product. Moreover, these conditions can cause the formation of antibody aggregates that can be immunogenic, so that careful monitoring is required for process validation.
In addition, repeated cycles of binding and elution, together with periodic cleaning and sanitization of the resin with 0.1-1N NaOH, can denature the tertiary structures of Protein A and Protein G, thereby decreasing the binding affinity and resin lifetime, and causing the release of immunogenic leachates in the mainstream. Lowe, C. R. (2001) Combinatorial approaches to affinity chromatography, Curr. Opin. Chem. Biol. 5, 248-256. D. K. Follman, R. L. Fahmer, J. Chromatogr. A 1024 (2004) 79; G. Hale, A. Drumm, P. Harrison, J. Phillips, J. Immunol. Methods 171 (1994), 15; T. Ishihara, T. Kadoya, N. Endo, S. Yamamoto, J. Chromatogr. A 1114 (2006) 97; P. Gagnon, Purification Tools for Monoclonal Antibodies, Validated Biosystems (1996); J. W. Bloom, M. F. Wong, G. Mitra, J. Immunol. Methods 117 (1989) 83; K. Brorson, J. Brown, E. Hamilton, K. E. Stein, J. Chromatogr. A 989 (2003) 115. Although engineered versions of Protein A and Protein G for greater stability exist, they are also more expensive. Furthermore, the above discussed issues of stability and harsh elution conditions remain a concern. Gulich S., Linhult M., Stahl S., and Hober S. (2002) Engineering streptococcal Protein G for increased alkaline stability, Protein Eng. 15, 835-842; Linhult M., Gulich S., Graslund T., Simon A., Karlsson M., Sjoberg A., Nord K., and Hober S. (2004) Improving the tolerance of a Protein A analogue to repeated alkaline exposures using a bypass mutagenesis approach, Proteins, 55, 407-416.
To address these concerns, academia and industry have tried to develop synthetic, efficient and less costly affinity ligands for antibodies. Some of these have been well characterized and even commercialized, such as the (i) hydrophobic charge induced ligand MEP (4-mercapto ethyl pyridine) marketed as BioSepra® MEP HyperCel® (Boschetti E. (2001) The use of thiophilic chromatography for antibody purification: a review, J. Biochem. Biophys. Methods 49, 361-389; Schwartz W., Judd D., Wysocki M., Guerrier L., Birck-Wilson E. and Boschetti E. (2001) Comparison of hydrophobic charge induction chromatography with affinity chromatography on Protein A for harvest and purification of antibodies. J. Chromatogr., A 908, 251-263; Boschetti E. (2002) Antibody separation by hydrophobic charge induction chromatography, Trends Biotechnol. 20, 333-337; Guerrier L., Flayeux I., and Boschetti E. (2001) A dual-mode approach to the selective separation of antibodies and their fragments, J. Chromatogr. B. 755, 37-46; and Guerrier L., Girot P., Schwartz W., and Boschetti E. (2000) New method for the selective capture of antibodies under physiological conditions, Bioseparation 9, 211-2210), (ii) the Protein A mimetic peptide Kaptiv-GY based on the sequence (RTY)4K2KG (TG19318) (Verdoliva A., Pannone F., Rossi M., Catello S., and Manfredi V. (2002) Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with Protein A and Protein G, J. Immunol. Methods 271, 77-88; Fassina G., Verdoliva A., Palombo G., Ruvo M., and Cassani G. (1998) Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody affinity purification, J. Mol. Recognit. 11, 128-133; and Fassina G., Ruvo M., Palombo G., Verdoliva A., and Marino M. (2001) Novel ligands for the affinity-chromatographic purification of antibodies, J. Biochem. Biophys. Methods 49, 481-490), (iii) the mixed-mode chromatographic ligand FastMabs A (Lihme A., and Hansen M. B. (2002) Isolation of immunoglobulins, Upfront Chromatography A/S (Copenhagen, DK); and Hansen M. B., Lihme A., Spitali M., and King D. (1998) Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent, Bioseparation 8, 189-193), and (iv) the MAbSorbent A2P derived from a triazine derivative 22/8 (Teng S. F., Sproule K., Husain A., and Lowe C. R. (2000) Affinity chromatography on immobilized “biomimetic” ligands synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand, J. Chromatogr. B. 740, 1-15; and Newcombe A. R., Cresswell C., Davies S., Watson K., Harris G., O'Donovan K., and Francis R. (2005) Optimized affinity purification of polyclonal antibodies from hyper immunized ovine serum using a synthetic Protein A adsorbent, MAbSorbent® A2P, J. Chromatogr. B. Biomed. Sci. Appl. 814, 209-215). Nevertheless, these small ligands have not replaced much of the market for Protein A affinity chromatography, primarily due to their lack of specificity, and the search for new ligands to be used in antibody purification is still quite intensive in both industry and academia. Feng H. Q., Jia L. Y., Li H. L., and Wang X. C. (2006) Screening and chromatographic assessing of a novel IgG biomimetic ligand, Biomed. Chromatogr. 20, 1109-1115. Specifically, attempts have been made over the last two decades to develop specific, chemically robust and cost-effective synthetic ligands. K. Sproule, P. Morrill, J. C. Pearson, S. J. Burton, K. R. Hejnaes, H. Valore, S. Ludvigsen, C. R. Lowe, J. Chromatogr. B 740 (2000) 17; A. C. A. Roque, C. S. O, Silva, M. A. Taipa, J. Chromatogr. A 1160 (2007) 44; C. R. Lowe, Curr. Opin. Chem. Biol. 5 (2001) 248. In particular, peptide ligands are of great interest due to their high specificity and stability and low cost compared to protein ligands.
Several peptide ligands for the purification of biomolecules from complex media have been identified. P. D. Bastek, J. M. Land, G. A. Baumbach, D. H. Hammond, R. G. Carbonell, Separation Sci. Technol. 35 (2000) 1681; P. V. Gurgel, R. G. Carbonell, H. E. Swaisgood, Sep. Sci. Technol. 36 (2001) 2411; G. Q. Wang, J. De, J. S. Schoeniger, D. C. Roe, R. G. Carbonell, J. Pept. Res. 64 (2004) 51; D. B. Kaufman, M. E. Hentsch, G. A. Baumbach, J. A. Buettner, C. A. Dadd, P. Y. Huang, D. H. Hammond, R. G. Carbonell, Biotechnol. Bioeng. 77 (2002) 278; C. L. Heldt, P. V. Gurgel, L. Jaykus and R. G. Carbonell, Biotechnol. Prog. 24 (2008) 554. Three linear hexapeptide ligands HWRGWV, HYFKFD and HFRRHL (SEQ ID NO: 1-3) were discovered that bind human IgG through its Fc portion, thus mimicking the binding mechanism of Protein A. H. Yang, P. V. Gurgel, R. G. Carbonell, J. Pept. Res. 66 (2005) 120; H. Yang, P. V. Gurgel, and R. G. Carbonell, J. Chromatogr. A 1216 (2009) 910. These hexapeptide ligands capture monoclonal and polyclonal antibodies from diverse sources demonstrating performances comparable to those offered by Protein A and Protein G. A. D. Naik, S. Menegatti, P. V. Gurgel, and R. G. Carbonell, J. Chromatogr. A 1218 (2011) 1691.
In a separate study, MS analysis of protease digests of the Fc fragment of hIgG revealed a putative binding sequence for HWRGWV (SEQ ID NO: 1) on the pFc segment which was found to be distinct from the Protein A and Protein G binding sites. Yang H., Gurgel P. V., Williams K., Bobay B., Cavanagh J., Muddiman D., and Carbonell R. G. (2009) Binding site on human immunoglobulin G for affinity ligand HWRGWV, J. Mol. Recognit., 23, 271-282. This result was consistent with the observation that the peptide HWRGWV (SEQ ID NO: 1) did not compete with either Protein A or Protein G for hIgG binding. Recent work has demonstrated that the ligand HWRGWV (SEQ ID NO: 1) is able to capture and purify commercial chimeric and humanized antibodies from industrial mammalian cell culture supernatants that include antifoam, host cell proteins, DNA, RNA, vitamins and many other contaminants. The antibody concentration of these cell cultures was slightly above 1 g/L and the yield and purity upon elution from the HWRGWV (SEQ ID NO: 1) column were 80% and 97% respectively. Also, the ligand was able to achieve two logs reduction of host cell protein and DNA. These results were similar to those obtained with Protein G column A. D. Naik, S. Menegatti, P. V. Gurgel, and R. G. Carbonell, J. Chromatogr. A 1218 (2011) 1691. HWRGWV-Toyopearl affinity resin has a capacity of 20 g/L (Yang H. Gurgel P. V., and Carbonell R. G. (2009)) and can be cleaned in acid and denaturing conditions. However, experimental work has shown that these adsorbents do not withstand the alkaline conditions (0.1 M-0.5 M NaOH) used in CIP and SIP treatments. In fact, upon exposure to alkaline wash the peptide ligand is leached from the adsorbent, resulting in a significant loss of binding capacity.
Accordingly, there is an important commercial need for alkaline-stable affinity chromatography reagents.